164 Methods of Observing Bacteria 



Nicolle* suggests the following modification of the technic : 



a. For Cover-glass Specimens: 



1. Stain for one to five minutes in a warm solution made as fol- 



lows: 10 c.c. of saturated alcoholic solution of gentian violet, 

 100 c.c. of a i per cent, aqueous solution of carbolic acid. 



2. Immerse from four to six seconds in the iodine-iodide of potas- 



sium solution. 



3. Decolorize in a mixture of 3 parts of absolute alcohol and i 



part of acetone. 



4. Counter stain if desired. 



b. For Sections: 



1. Stain the nuclear elements of the tissue with carmine. For 



this Nicolle prefers Orth's carmine solution (5 parts of Orth's 

 carmine with i part of 95 per cent, alcohol). 



2. Stain in the carbol -gentian violet, as indicated above. 



3. Immerse for four to six seconds in the iodine-iodide of potas- 



sium solution. 



4. Differentiate with absolute alcohol containing 0.33 per cent. 



(by volume) of acetone. 



5. Treat with 95 per cent, alcohol containing some picric acid 



until the tissue is greenish yellow (one to five seconds). 



6. Dehydrate with absolute alcohol. 



7. Clear with xylol or other appropriate reagent. 



8. Mount in balsam. 



The Gram-Weigert Stain can be employed with beautiful 

 results for staining many micro-organisms. It differs from 

 the Gram method in that anilin oil instead of alcohol is 

 used for decolorizing. To secure the most brilliant results 

 it is best first to stain the tissue with alum, borax, or 

 lithium carmin, and then 



1. Stain in Ehrlich's anilin-oil-water gentian violet, 



five to twenty minutes; 



2. Wash off excess with normal salt solution; 



3. Immerse in dilute iodin solution (iodin i, iodid of 



potassium 2, water 100) for one minute; 



4. Drain off the fluid and blot the section spread out 



upon the slide, with absorbent paper; 



5. Decolorize with a mixture of equal parts of anilin 



and xylol; 



6. Wash out the anilin with pure xylol; 



7. Mount in xylol balsam. 



Eosin and Methylene-blue (Mallory) make a beautiful 

 contrast tissue stain for routine work, and also, demonstrates 



* "Ann. de 1'Inst. Pasteur," 1895, ix. 



